The proposed project relies on previous structural knowledge of avian Kir2.2 and bacterial Kir channels to probe the molecular details of ROMK (Kir1.1) channel gating. This continues the original specific goal of understanding potassium (K) permeation and gating through the renal, inward rectifying, K channel ROMK (Kir1.1); which plays an important role in control of systemic K and water balance. Results of this study would not only be relevant for renal diseases like antenatal Bartter's syndrome but might also be important for hypertension if a partial reduction of ROMK function lowers blood pressure without significantly disrupting serum electrolytes. Starting with crystallographic models of the avian Kir2.2 and prokaryotic KirBac closed-states, the proposed experiments would examine conformational changes associated with Kir1.1 channel gating (opening & closing), using direct measurement of state-dependent molecular distances with lanthanide resonance energy transfer (LRET) optical techniques in an in vitro proteoliposome system. AIM 1 describes steady-state LRET determinations of Kir1.1b dimensions using novel single-Cys dimeric constructs, labeled with a single donor and a single acceptor. In this aim we would also conduct electrophysiological measurements to validate these single-Cys dimers as models for ROMK gating. AIM 2 proposes state-dependent LRET measurements to evaluate alternative hypotheses for initiation of channel opening by the C-terminal domain; and AIM 3 examines alternative motions of the primary hydrophobic gate at the ROMK bundle-crossing of inner transmembrane helices. This would help resolve the Kir open-state conformation, which has been controversial. Finally, in AIM 4 we would evaluate the hypothesis that PIP2 binding, required for ROMK opening, sets a pre-open condition by shortening the linker between the C-terminal domain and the interfacial slide helix. The proposed expts rely on a variety of innovations: (1) cell- free, in vitro, eukaryotic protein expression, (2) evaluation of cell-free Kr protein by direct injection into Xenopus oocytes, followed by whole-cell and excised patch recording (3) unambiguous LRET state-dependent molecular distance measurements using single-Cys ROMK dimers.